Activating mutations in BRAF and KRAS, key signaling components of the MAPK pathway, are among the most common oncogenic mutations in human cancer, occurring in ~7% and ~20% of all cancers and in ~10-15% and ~40% of colorectal cancers (CRCs), respectively. Consequently, effective targeted therapy strategies for cancers harboring BRAF and KRAS mutations would have a profound clinical impact, and inhibitors of MAPK signaling, including BRAF and MEK inhibitors, are currently in clinical development. While BRAF inhibitors proved remarkably effective in BRAF mutant melanoma, results have been disappointing in BRAF mutant CRC. Although the reason for this disparity is not fully understood, recent work from our group and others has found that EGFR signaling may play a key role in resistance. Our preliminary studies have also shown that BRAF inhibitor-based targeted therapy combinations, such as combined BRAF/MEK (currently in clinical trials) or combined BRAF/EGFR inhibition, may improve efficacy in BRAF mutant CRC. Although KRAS is the most commonly mutated oncogene, no effective therapies exist for KRAS mutant cancers, largely because the KRAS protein itself has proven difficult to target directly with small molecules. Targeting single downstream KRAS effector pathways (e.g. MEK) has also met limited success in clinical trials, likely because KRAS activates multiple important signaling pathways. Previously, our laboratory and others showed that combined targeting of more than one KRAS effector pathway (MEK and PI3K) can cause dramatic responses in KRAS mutant mouse tumor models, but preliminary data suggests that combined MEK/PI3K inhibition may only be effective in a subset of KRAS mutant cancers, underscoring the need for additional effective inhibitor combinations. The overall hypothesis of this proposal is that the limited efficacy of BRAF and MEK inhibitors in BRAF and KRAS mutant CRC, respectively, involves activation of other key pathways that reduce the extent to which these cancers are addicted to MAPK signaling, constraining the effectiveness of these inhibitors as single-agents. We aim to develop novel combination therapy strategies for BRAF and KRAS mutant cancers, focusing specifically on CRC. Building on our recent discovery that EGFR mediates resistance to BRAF inhibitors in BRAF mutant CRC, we will perform a comprehensive analysis of the role of EGFR in BRAF inhibitor resistance, utilizing in vitro models and patient tumor biopsies from a novel targeted therapy trial for BRAF mutant CRC. In KRAS mutant CRC, we will employ a novel pooled shRNA drug screen developed in our laboratory to identify new targets for combination therapy with MEK inhibitors. Novel combinations will be evaluated in cutting-edge mouse models of KRAS mutant CRC with the goal of rapidly identifying effective targeted therapy combinations. New therapeutic strategies for these cancers would represent a major advance in personalized cancer medicine with significant clinical benefit for a large population of cancer patients.